As a result of the sequencing of the human genome, it has become apparent that the complexity of an organism does not necessarily correlate with the size of its genome. This has sparked an interest in discovering exactly how genes are regulated on levels other than primary genomic sequence. Epigenetics has arisen as a field that addresses this concern by focusing on post-transcriptional methods of gene expression control including DNA methylation, histone post-translational modifications (PTMs) and non-coding RNAs.1
Modifications made to proteins post-translationally can affect their function, location or longevity. One set of highly modified proteins important to gene expression is histones. DNA is wrapped around histones, which form nucleosomes before being condensed into chromatin and ultimately chromosomes.
Histone PTMs have been shown to be very important to gene expression. Some modifications serve to signal the recruitment of chromatin modifying enzymes while some serve to alter the interaction between the histones and DNA allowing or prohibiting the access of transcription machinery.2
Some more common histone modifications include methylation, acetylation and phosphorylation. Of these common modifications, methylation is by far the most complex. Both lysine and arginine residues can be modified by mono- or di-methylation and lysine residues can be tri-methylated as well. These varying states of methylation can be associated with both active and inactive genes. The complexity and importance of methylation, on histones in particular, has stirred much interest in the enzymes capable of adding (methyltransferases) and removing (demethylases) these modifications.
The first demethylase was discovered in 2004 and was termed Lysine Specific Demethylase 1 or LSD1.3
LSD1 is a flavin-dependent amine oxidase that can remove mono- and di-methyl marks from H3K4 primarily, H3K9 under certain conditions and some non-histone substrates such as p53.4–6
It has been shown to be part of many protein complexes including CoREST, NuRD and AR/ER.7
LSD1 is also associated with gene repression and has been suggested to be important in initiating myc-induced transcription in cancers.3,8–11
Structural and biochemical studies have led to the development of numerous LSD1 inactivators that have the potential to be keen therapeutic tools, much like the successful deacetylase inhibitors currently in use.12
In addition, the mechanism of LSD1 indicates that it is an excellent candidate for suicide inactivators. Many monoamine oxidase (MAO) inhibitors have been suggested as potential LSD1 suicide inactivators.13
Several different assays are used to study the activity of LSD1 in the presence and absence of these various inhibitors in order to determine their efficiency.
Currently, histone demethylase activity can be accurately measured using a handful of assays. One assay involves synthetic methylation of histone substrates using a recombinant methyltransferase and isotopically heavy S-adenosyl methionine.14
The removal of tritiated methyl groups from histone substrates is monitored by autoradiography. Also, due to the formation of formaldehyde in certain demethylase reactions, formaldehyde release can be followed to determine demethylase activity. While these methods are useful in determining if an enzyme is acting as a demethylase, they are not quantitative in nature. Another common method for following demethylase activity is western blotting using antibodies specific to certain modifications.15
While western blotting can be used as a semi-quantitative technique, the antibodies used for detection of histone modifications are often non-specific making absolute quantification difficult.16
Using antibody-based methods to quantify different methyl states of a single residue is especially difficult due to the similarity in structure of mono-, di- and tri-methylated lysines.17
As an alternative, mass spectrometry is often used as a tool for studying the activity of demethylases on synthetic peptides.18
This technique provides a rapid method for highly sensitive analysis of full products of demethylase reactions. One caveat to using mass spectrometry for such an analysis is that it is, at best, semi-quantitative because differentially modified peptides cannot be equally compared. For example, a mono-methylated peptide will not necessarily ionize the same as a tri-methylated species of the exact same sequence. This leads to a difficulty in using mass spectrometry-based assays to quantify the efficiency of demethylases.
To address the issues of differential ionization and sample variation, it is common to use isotopically heavy chemistry to modify peptides. For example, H4 lysine acetylation has been quantified by using isotopically heavy acetic anhydride to synthetically acetylate all lysine residues, and recent applications of this approach have incorporated MALDI mass spectrometers, allowing for more straightforward data analysis.19,20
We present an assay which employs reductive methylation, using deuterated formaldehyde, followed by MADLI mass spectrometry, to differentiate between various lysine methylation states. This method accounts for methyl states within a sample, allowing sample-to-sample relative comparison.
Reductive methylation is commonly used as a method to improve the stability of proteins for analysis by x-ray crystallography.21
During this process, lysine residues are chemically di-methylated using the following reactions to alkylate and then reduce amines:
In this paper, we use a combination of reductive methylation, isotopic labeling and mass spectrometry to quantitatively measure the activity of LSD1 alone and in the presence of an MAO inactivator. We have termed our assay MassSQUIRM (Mass Spectrometric Quantitation Using Isotopic Reductive Methylation). MassSQUIRM will prove invaluable to the further study of the mechanism of not only LSD1 but also many methyltransferase and demethylase enzymes and the efficiency of their proposed inhibitors. Effective LSD1 inactivators have the potential to become targeted therapies with high specificity that could potentially treat LSD1-associated diseases, such as cancer, with minimal side effects.